Coryneform bacteria are traditionally industrial microorganisms which are most widely used for the production of a variety of chemical materials useful in the animal feed, medicine and food industries, including amino acids, such as L-lysine, L-threonine, L-arginine, L-threonine and glutamic acid, and nucleic acid-related materials. These microorganisms are Gram-positive and require biotin for their growth. They are divided by snapping and their poor ability to degrade the metabolites they produce can be advantageously utilized. Representative examples of coryneform bacteria include Corynebacterium genus, such as Corynebacterium glutamicum, Brevibacterium genus, such as Brevibacterium flavum, Athrobacter spp. and Microbacterium spp. etc.
L-lysine is a commercially important L-amino acid which is used as a feed additive in animal nutrition thanks to its ability to help the body to absorb other amino acids thus improving the quality of the feedstuff. For the body, L-lysine is used as an ingredient of an injection solution, and also finds applications in the pharmaceutical field. Therefore, the industrial production of L-lysine is economically important industrial process.
The production yield of lysine is correlated with enzyme activity on the biosynthesis pathway which can be typically enhanced by amplifying one or more genes on the biosynthesis pathway of lysine or by employing a modified promoter for the genes. Corynebacterium strains with lysine biosynthesis-associated genes enhanced therein and the production of L-lysine using the same are well known. For example, U.S. Pat. No. 6,746,855 discloses a process for the production of L-lysine by fermenting an L-lysine producing corynebacteria with enhanced lysE gene (lysine export carrier gene), in which additionally genes selected from the group consisting of a dapA gene, an lysC gene, a pyc gene and a dapB gene are enhanced. U.S. Pat. No. 6,221,636 discloses corynebacteria transformed with a recombinant DNA comprising a nucleotide sequence coding for an aspartokinase in which feedback inhibition by L-lysine and L-threonine is substantially desensitized and a nucleotide sequence coding for a diaminopimelate decarboxylase.
Korean Patent No. 10-0345592 describes an Escherichia strain into which dapA and lysC, both modified to eliminate the feedback inhibition by L-lysine, are introduced and in which dapB and ddh genes are amplified, and a method for the production of L-lysine using the same.
For the development of coryneform bacteria into variants capable of producing target products at high titers, a genetic or metabolic engineering technique by which genes involved in the metabolism can be selectively controlled is needed. To this end, it is important to modify a promoter activity, a regulatory DNA region which provides a secure initial binding site for RNA polymerase to control the transcription of regulated genes.
Modified promoters originating from coryneform bacteria are found in many patents. For example, promoters from corynebacterium are described in U.S. Pat. No. 5,700,661 entitled “Gene expression regulatory DNA”, U.S. Pat. No. 5,965,391 entitled “DNA which regulates gene expression in coryneform bacteria”, U.S. Pat. No. entitled 7,141,388 “Nucleotide sequences for transcriptional regulation in corynebacterium glutamicum”, and Korean Patent No. 10-0653742 entitled “Novel L-lysine-inducible promoter”, and a promoter from Corynebacterium ammoniagenes is described in Korean Patent Publication No. 10-2006-0068505 entitled “Novel promoter nucleic acid originating from corynebacterium genus bacteria, expression cassette comprising the promoter and vector comprising the cassette, host cell comprising the vector and method for expressing a gene using the cell”.
However, none of the coryneform bacteria which are improved in the activity of ddh (diaminopimelate dehydrogenase) which plays a critical role in the lysine biosynthesis pathway, with an enhanced promoter for the enzyme substituted for the endogenous one on the genome of the host cell have been disclosed thus far.